Recombinant Rat TNF-α

Recombinant Rat TNF-α is widely used in research applications because it is a key cytokine involved in regulating inflammation, immune responses, apoptosis, and cell signaling, making it essential for modeling disease mechanisms and testing therapeutic interventions in rat-based systems.

Key scientific reasons to use recombinant rat TNF-α:

• Species-specific activity: Using rat TNF-α ensures physiological relevance in rat models, as cytokine-receptor interactions can be species-specific, affecting experimental outcomes.
• Central role in inflammation and immunity: TNF-α is a pleiotropic molecule that orchestrates inflammatory responses, immune cell regulation, and apoptosis, making it crucial for studies on autoimmune diseases, infection, and tissue injury.
• Disease modeling: Recombinant rat TNF-α is used to induce or modulate disease states such as rheumatoid arthritis, intervertebral disc degeneration, and neuropathic pain in vivo, allowing for mechanistic studies and drug testing.
• Cellular and molecular assays: It is applied in bioassays to study cytokine signaling, cell death, and immune cell activation in vitro, facilitating the dissection of TNF-α pathways and their modulation by drugs or genetic interventions.
• Therapeutic development: Recombinant TNF-α is used to screen and validate anti-TNF therapies, investigate receptor selectivity, and explore novel drug delivery systems, supporting translational research.
• Standardization and reproducibility: Recombinant proteins provide consistent, defined activity and purity, enabling reproducible results across experiments and laboratories.

Typical applications include:

• Inducing inflammatory responses in cell cultures or animal models.
• Studying cytokine-mediated apoptosis and necrosis.
• Investigating TNF-α’s role in osteoclastogenesis, metabolic regulation, and tumor biology.
• Testing anti-inflammatory or immunomodulatory compounds targeting TNF-α signaling.

Best practices:

• Use recombinant rat TNF-α for rat cell lines or in vivo rat studies to ensure accurate receptor binding and downstream signaling.
• Validate activity and endotoxin levels to avoid confounding immune responses.
• Optimize dosing and timing based on experimental goals and published protocols.

In summary, recombinant rat TNF-α is indispensable for immunology, inflammation, and disease research in rat models, providing species-specific, reproducible, and mechanistically relevant insights into TNF-α biology and therapeutic targeting.

Yes, recombinant rat TNF-α can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated and matched to your assay system. Recombinant TNF-α is widely used as a standard in commercial rat TNF-α ELISA kits and in published protocols for quantitative measurement.

Supporting details:

• Specificity and Parallelism: ELISA kits designed for rat TNF-α typically recognize both natural and recombinant forms, and dose-response curves for recombinant TNF-α are parallel to those for natural TNF-α, indicating suitability for quantification.
• Calibration: Many ELISA kits are calibrated against highly purified recombinant rat TNF-α, and the standard curve generated with recombinant protein is used to interpolate sample concentrations.
• Applications: Recombinant rat TNF-α is routinely used as an immunoassay standard for building calibration curves in ELISA protocols measuring TNF-α in serum, plasma, cell culture supernatant, and tissue lysates.
• Validation: For best practice, ensure the recombinant TNF-α standard is validated for your specific ELISA system. Check that the standard curve generated with recombinant TNF-α is parallel to curves obtained with natural TNF-α in your sample matrix. This confirms that the assay quantifies both forms equivalently.

Best practices:

• Use recombinant TNF-α that matches the extracellular domain sequence of native rat TNF-α, as this is the immunoreactive portion detected by most ELISA antibodies.
• Prepare serial dilutions of the recombinant standard in the same buffer or diluent used for your samples to minimize matrix effects.
• Always run the standard curve in parallel with your samples for accurate quantification.

Limitations:

• Recombinant standards are for research use only and not for diagnostic procedures.
• Minor differences in glycosylation or folding between recombinant and native TNF-α (depending on the expression system) are generally not significant for ELISA quantification, but should be considered if absolute quantification is critical.

Summary Table: Recombinant Rat TNF-α as ELISA Standard

In conclusion, recombinant rat TNF-α is appropriate for use as a standard in ELISA quantification and calibration, provided it is validated for your assay system and used according to best practices.

Recombinant Rat TNF-α has been validated for a range of applications in published research, primarily in the context of inflammation, immune modulation, and cell signaling studies. The most commonly validated applications include:

• In Vivo Studies: Used to induce or modulate inflammatory responses, model disease states (such as rheumatoid arthritis, intervertebral disc degeneration, renal disease, neuropathic pain, and sleep regulation), and assess therapeutic interventions in rat models.
• Bioassays: Employed to measure biological activity, such as cytotoxicity (e.g., using L-929 fibroblast cells), cytokine induction, and signaling pathway activation in cultured cells.
• ELISA (Enzyme-Linked Immunosorbent Assay): Used as a standard or control for quantifying TNF-α levels in biological samples, particularly in infection and inflammation models.
• Immunomodulation and Gene Therapy Research: Utilized in studies investigating the effects of TNF-α antagonism or RNA interference (RNAi) strategies for diseases like rheumatoid arthritis, including oral delivery of TNF-α shRNA.
• Western Blotting and SDS-PAGE: Used as a positive control or standard for protein detection and quantification in biochemical assays.
• Mass Cytometry (CyTOF) and Spatial Biology: Applied in advanced cell profiling and tissue analysis techniques.

Supporting details and examples from published research:

• In Vivo Applications: Recombinant rat TNF-α has been used to induce inflammation in models of intervertebral disc degeneration, abdominal aortic aneurysm, renal disease, and neuropathic pain, as well as to study its effects on sleep regulation and neuronal sensitivity.
• Bioassays: Validated for use in cytotoxicity assays (e.g., L-929 mouse fibroblast cells), cytokine mRNA induction, and signaling pathway studies in whole cells.
• ELISA: Used as a standard in rat pneumonia and soft-tissue infection models to quantify TNF-α.
• Gene Therapy Research: Studies have used recombinant TNF-α and its antagonists to evaluate immunomodulatory effects and safety in rheumatoid arthritis models, including oral delivery of TNF-α shRNA.
• Protein Detection: Recombinant rat TNF-α is suitable for SDS-PAGE and Western blotting as a standard or control protein.

Summary Table of Validated Applications

These applications are supported by multiple peer-reviewed studies and product validation data, confirming the utility of recombinant rat TNF-α in both basic and translational research contexts.

To reconstitute and prepare Recombinant Rat TNF-α protein for cell culture experiments, dissolve the lyophilized protein in sterile buffer at a concentration of at least 100 μg/mL, using sterile PBS or distilled water, and include 0.1% serum albumin if possible to stabilize the protein.

Step-by-step protocol:

• Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
• Reconstitution buffer: Use sterile PBS or sterile distilled water. For enhanced stability, add at least 0.1% human or bovine serum albumin (BSA).
• Concentration: Do not reconstitute to less than 100 μg/mL. Typical working concentrations for cell culture range from 0.1–1.0 mg/mL for stock solutions.
• Mixing: Gently swirl or invert the vial to dissolve. Avoid vortexing or vigorous pipetting, which can denature the protein.
• Aliquoting: Divide the reconstituted solution into small aliquots in polypropylene tubes to minimize freeze-thaw cycles.
• Storage: Store aliquots at −20 °C to −70 °C. Avoid repeated freeze-thaw cycles, as this can reduce bioactivity.
• Dilution for cell culture: Before use, dilute the stock solution into cell culture medium to the desired final concentration. Ensure the medium is compatible with TNF-α and does not contain components that may inactivate the cytokine.

Additional notes:

• Always bring the protein to room temperature before use.
• For sensitive applications, filter-sterilize the final working solution if necessary.
• The presence of carrier protein (BSA) is recommended for long-term stability, especially at low concentrations.
• Avoid storing reconstituted protein at 2–8 °C for more than 1 month.

Summary Table:

This protocol ensures optimal solubility, stability, and bioactivity of recombinant rat TNF-α for cell culture experiments.